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Biochimica Et Biophysica Acta.... Aug 2023Adaptability to intracellular or extracellular cues is essential for maintaining cellular homeostasis. Metabolic signals intricately control the morphology and functions...
Adaptability to intracellular or extracellular cues is essential for maintaining cellular homeostasis. Metabolic signals intricately control the morphology and functions of mitochondria by regulating bioenergetics and metabolism. Here, we describe the involvement of PHLPP1, a Ser/Thr phosphatase, in mitochondrial homeostasis. Microscopic analysis showed the enhanced globular structure of mitochondria in PHLPP1-depleted HEK 293T and C2C12 cells, while forced expression of PHLPP1 promoted mitochondrial tubularity. We show that PHLPP1 promoted pro-fusion markers MFN2 and p-DRP1 levels using over-expression and knockdown strategies. Contrastingly, PHLPP1 induced mitochondrial fragmentation by augmenting pro-fission markers, t-DRP1 and pDrp1 upon mitochondrial stress. At the molecular level, PHLPP1 interacted with and caused dephosphorylation of calcineurin, a p-DRP1 phosphatase, under basal conditions. Likewise, PHLPP1 dimerized with PINK1 under basal conditions. However, the interaction of PHLPP1 with both calcineurin and PINK1 was impaired upon CCCP and oligomycin-induced mitochondrial stress. Interestingly, upon mitochondrial membrane depolarization, PHLPP1 promoted PINK1 stabilization and parkin recruitment to mitochondria, and thereby activated the mitophagy machinery providing a molecular explanation for the dual effects of PHLPP1 on mitochondria under different conditions. Consistent with our in-vitro findings, depletion of phlp-2, ortholog of PHLPP1 in C. elegans, led to mitochondrial fission under basal conditions, extended the lifespan of the worms, and enhanced survival of worms subjected to paraquat-induced oxidative stress.
Topics: Animals; Caenorhabditis elegans; Calcineurin; Longevity; Protein Kinases; Ubiquitin-Protein Ligases; HEK293 Cells; Humans; Mice
PubMed: 37060964
DOI: 10.1016/j.bbadis.2023.166718 -
Autophagy Nov 2023The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and...
The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy. AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type.
Topics: Animals; Mice; Autophagy; Leukemia, Myeloid, Acute; Mitophagy; Reactive Oxygen Species; Ubiquitin-Protein Ligases; Ubiquitins; Humans
PubMed: 37439113
DOI: 10.1080/15548627.2023.2230839 -
Life Science Alliance Feb 2024Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are...
Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here, we investigated whether and how cristae morphology and dynamics are dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨ), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. CCCP treatment leading to ΔΨ abrogation even enhanced cristae dynamics showing its ΔΨ-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.
Topics: Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Mitochondria; Mitochondrial Membranes; Oxidative Phosphorylation; Adenosine Triphosphate; Mammals
PubMed: 37957016
DOI: 10.26508/lsa.202302386 -
Life Sciences Jan 2024Mitochondria are important organelles in cells responsible for energy production and regulation. Mitochondrial dysfunction has been implicated in the pathogenesis of... (Review)
Review
Mitochondria are important organelles in cells responsible for energy production and regulation. Mitochondrial dysfunction has been implicated in the pathogenesis of many diseases. Oligomycin sensitivity-conferring protein (OSCP), a component of the inner mitochondrial membrane, has been studied for a long time. OSCP is a component of the F1Fo-ATP synthase in mitochondria and is closely related to the regulation of the mitochondrial permeability transition pore (mPTP). Studies have shown that OSCP plays an important role in cardiovascular disease, neurological disorders, and tumor development. This review summarizes the localization, structure, function, and regulatory mechanisms of OSCP and outlines its role in cardiovascular disease, neurological disease, and tumor development. In addition, this article reviews the research on the interaction between OSCP and mPTP. Finally, the article suggests future research directions, including further exploration of the mechanism of action of OSCP, the interaction between OSCP and other proteins and signaling pathways, and the development of new treatment strategies for mitochondrial dysfunction. In conclusion, in-depth research on OSCP will help to elucidate its importance in cell function and disease and provide new ideas for the treatment and prevention of related diseases.
Topics: Humans; Adenosine Triphosphatases; Carrier Proteins; Cardiovascular Diseases; Mitochondrial Proton-Translocating ATPases; Mitochondrial Diseases; Neoplasms
PubMed: 38030056
DOI: 10.1016/j.lfs.2023.122293 -
Theriogenology Oct 2023Cellular metabolism is an important feature of spermatozoa that deserves more insights to be fully understood, in particular in porcine semen physiology. The present...
Cellular metabolism is an important feature of spermatozoa that deserves more insights to be fully understood, in particular in porcine semen physiology. The present study aims to characterize the balance between glycolytic and oxidative metabolism in boar sperm cells. Agilent Seahorse technology was used to assess both oxygen consumption rate (OCR), as an oxidative metabolism index, and extracellular acidification rate (ECAR), as a glycolytic index. Different metabolic parameters were studied on freshly ejaculated sperm cells (identified as day zero sample, d0) and after one day of storage at 17 °C in Androhep extender (d1). Mitochondrial ATP production rate (MitoATP) was higher than the glycolytic ATP production rate (glycoATP) at both d0 and d1 while at d1 the amount of ATP production decreased, in particular, due to OXPHOS reduction. Conversely, glycoATP was not significantly different between d0 and d1. Interestingly, OCR profile showed no different bioenergetic parameters (i.e. ATP turnover, basal or maximal respiration, and spare respiration) between d0 and d1, thus indicating that sperm cell metabolism was reversibly decreased by preservation conditions. Other metabolic parameters showed the same trend, irrespective of the storage time: under stressed conditions (oligomycin plus FCCP), spermatozoa showed an increase in mitochondrial respiration while the metabolic potential of glycolysis did not undergo variations when compared to baseline metabolism. The rate of oxidation of fuel substrates - glucose, fatty acids, and glutamine - showed that sperm reliance on glucose oxidation to maintain baseline respiration was higher than fatty acids or glutamine. Interestingly spermatozoa demonstrated to have a low "capacity" parameter, which indicates that they cannot use only a single fuel substrate to produce energy. This feature of sperm metabolism to be unable to increase oxidation of a particular fuel to compensate for inhibition of alternative fuel pathway(s) was demonstrated by the negative value of "flexibility". Our results showed that ATP production in boar sperm cells relied on mitochondrial oxidative metabolism in freshly ejaculated cells, while, under liquid storage conditions, their oxidative metabolism decreased while the glycolysis remained constant. These results open new fields of research in the preservation techniques of boar sperm cells.
Topics: Male; Animals; Swine; Glutamine; Semen; Energy Metabolism; Spermatozoa; Glucose; Adenosine Triphosphate
PubMed: 37517301
DOI: 10.1016/j.theriogenology.2023.07.018 -
BMC Anesthesiology Sep 2023Dexamethasone (Dexa) has been recently found to exert an analgesic effect, whose action is closely related to IL-8. However, whether dexamethasone induces...
BACKGROUND
Dexamethasone (Dexa) has been recently found to exert an analgesic effect, whose action is closely related to IL-8. However, whether dexamethasone induces antinociception via glycolysis and mitochondria-related pathways is still unclear.
METHODS
Right hind paw inflammatory pain in mice was induced by intraplantar injection of Freund's Complete Adjuvant (FCA). Von Frey test was then used to measure the paw withdrawal threshold. The detection of glycolysis and mitochondrial pathway-related proteins and IL-8 were determined by Western blot and ELISA. The potential interaction between Dexa and fructose-1,6-bisphosphate (FBP, a PKM2 activator) was examined by simulation predictions using molecular docking.
RESULTS
Intrathecal administration of Dexa (20 µg/20 µL) had an obvious analgesic effect in FCA-treated mice, which was counteracted by the glycolysis inhibitor 2-deoxyglucose (2-DG, 5 mg/20 µL) or the mitochondria-related pathway inhibitor oligomycin complex (Oligo, 5 µg/20 µL). In the glycolysis pathway, Dexa decreased GLUT3 and had no impact on HIF-1α expression during FCA-induced inflammation. Additionally, Dexa further increased the PKM2 level, accompanied by the formation of hydrogen bonds between Dexa and the PKM2 activator fructose-1,6-bisphosphate (FBP). In the mitochondrial pathway, Dexa downregulated the expression of Mfn2 protein but not the PGC-1α and SIRT-1 levels in the spinal cord. Moreover, both 2-DG and Oligo decreased Mfn2 expression. Finally, IL-8 level was reduced by the single or combined administration of Dexa, 2-DG, and Oligo.
CONCLUSION
Dexa attenuated IL-8 expression via glycolysis and mitochondrial pathway-related proteins, thus mediating the analgesic effect during inflammatory pain.
Topics: Animals; Mice; Interleukin-8; Molecular Docking Simulation; Fructose; Glycolysis; Mitochondria; Dexamethasone; Analgesics
PubMed: 37723417
DOI: 10.1186/s12871-023-02277-9 -
Stem Cell Research & Therapy Nov 2023The metabolic reprogramming of mesenchymal stem/stromal cells (MSC) favoring glycolysis has recently emerged as a new approach to improve their immunotherapeutic...
BACKGROUND
The metabolic reprogramming of mesenchymal stem/stromal cells (MSC) favoring glycolysis has recently emerged as a new approach to improve their immunotherapeutic abilities. This strategy is associated with greater lactate release, and interestingly, recent studies have proposed lactate as a functional suppressive molecule, changing the old paradigm of lactate as a waste product. Therefore, we evaluated the role of lactate as an alternative mediator of MSC immunosuppressive properties and its contribution to the enhanced immunoregulatory activity of glycolytic MSCs.
MATERIALS AND METHODS
Murine CD4 T cells from C57BL/6 male mice were differentiated into proinflammatory Th1 or Th17 cells and cultured with either L-lactate, MSCs pretreated or not with the glycolytic inductor, oligomycin, and MSCs pretreated or not with a chemical inhibitor of lactate dehydrogenase A (LDHA), galloflavin or LDH siRNA to prevent lactate production. Additionally, we validated our results using human umbilical cord-derived MSCs (UC-MSCs) in a murine model of delayed type 1 hypersensitivity (DTH).
RESULTS
Our results showed that 50 mM of exogenous L-lactate inhibited the proliferation rate and phenotype of CD4 T cell-derived Th1 or Th17 by 40% and 60%, respectively. Moreover, the suppressive activity of both glycolytic and basal MSCs was impaired when LDH activity was reduced. Likewise, in the DTH inflammation model, lactate production was required for MSC anti-inflammatory activity. This lactate dependent-immunosuppressive mechanism was confirmed in UC-MSCs through the inhibition of LDH, which significantly decreased their capacity to control proliferation of activated CD4 and CD8 human T cells by 30%.
CONCLUSION
These findings identify a new MSC immunosuppressive pathway that is independent of the classical suppressive mechanism and demonstrated that the enhanced suppressive and therapeutic abilities of glycolytic MSCs depend at least in part on lactate production.
Topics: Humans; Male; Animals; Mice; Mice, Inbred C57BL; Lactic Acid; Immunosuppressive Agents; Mesenchymal Stem Cells; Cell Differentiation
PubMed: 37981698
DOI: 10.1186/s13287-023-03549-4 -
BMC Cancer Nov 2023Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse...
Synergistic lethality in chronic myeloid leukemia - targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment.
Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.
Topics: Humans; Tyrosine Kinase Inhibitors; Fusion Proteins, bcr-abl; Oxidative Phosphorylation; Thapsigargin; Drug Resistance, Neoplasm; Protein Kinase Inhibitors; Enzyme Inhibitors; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Oligomycins; Adenosine Triphosphate; Apoptosis
PubMed: 38012567
DOI: 10.1186/s12885-023-11623-6 -
International Journal of Experimental... Dec 2023This study aimed to investigate the effects of mitochondrial homeostasis on lipopolysaccharide (LPS)-induced endothelial cell barrier function and the mechanisms that...
This study aimed to investigate the effects of mitochondrial homeostasis on lipopolysaccharide (LPS)-induced endothelial cell barrier function and the mechanisms that underlie these effects. Cells were treated with LPS or oligomycin (mitochondrial adenosine triphosphate synthase inhibitor) and the mitochondrial morphology, mitochondrial reactive oxygen species (mtROS), and mitochondrial membrane potential (ΔΨm) were evaluated. Moreover, the shedding of glycocalyx-heparan sulphate (HS), the levels of HS-specific degrading enzyme heparanase (HPA), and the expression of occludin and zonula occludens (ZO-1) of Tight Junctions (TJ)s, which are mediated by myosin light chain phosphorylation (p-MLC), were assessed. Examining the changes in mitochondrial homeostasis showed that adding heparinase III, which is an exogenous HPA, can destroy the integrity of glycocalyx. LPS simultaneously increased mitochondrial swelling, mtROS, and ΔΨm. Without oligomycin effects, HS, HPA levels, and p-MLC were found to be elevated, and the destruction of occludin and ZO-1 increased. Heparinase III not only damaged the glycocalyx by increasing HS shedding but also increased mitochondrial swelling and mtROS and decreased ΔΨm. Mitochondrial homeostasis is involved in LPS-induced endothelial cell barrier dysfunction by aggravating HPA and p-MLC levels. In turn, the integrated glycocalyx protects mitochondrial homeostasis.
Topics: Lipopolysaccharides; Occludin; Endothelial Cells; Tight Junctions; Oligomycins
PubMed: 37828780
DOI: 10.1111/iep.12495 -
MSphere Aug 2023is a prominent opportunistic fungal pathogen of humans. The increasing incidence of infections is attributed to both innate and acquired resistance to antifungals....
is a prominent opportunistic fungal pathogen of humans. The increasing incidence of infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (, , , , ). A bZIP transcription repressor of mitochondrial function () positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in . Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.
Topics: Humans; Antifungal Agents; Candida glabrata; Cycloheximide; Drug Resistance, Fungal; Fluconazole; Fungal Proteins; Transcription Factors; Xenobiotics
PubMed: 37358297
DOI: 10.1128/msphere.00254-23